The use of immobilized enzymes, i.e., enzymes bound to a solid carrier, in continuously operated reactors is an increasingly preferred technique, since it enables savings in enzyme costs as well as in the purification of the final product. The conversion of glucose to fructose with an immobilized glucose isomerase is a process that commonly utilizes such a procedure.
In general, enzymes are water-soluble, thus making it necessary to use an immobilization technique in a continuous process. The enzyme has to be bound to a solid phase by a method that prevents it from dissolving in the aqueous phase while allowing it to maintain its activity. Various techniques have been suggested to associate enzymes with solid carriers wherein the enzyme is absorbed, covalently linked, cross-linked or microencapsulated. Alternatively, the entire microorganism producing the enzyme can be bound to a solid phase. A good summary of such techniques is presented in e.g. Moo-Young, M. (ed), Comprehensive Biotechnology, 2, Pergamon Press, London 1985, p. 191-211.
In prior processes, the enzyme is bound to a carrier material prepared separately, which as such may be advantageous to the chemical kinetics or the flow technique of the substrate. The carrier material is, however, in most cases more costly than the enzyme acting as a catalyst, especially in large-scale mass production processes, such as those used in the sugar industries. Alternatively, the enzyme can be immobilized by cross-linking it with an inert component, such as gelatin. In any case, the enzyme acting as a catalyst in the prior art forms only a fraction, generally less than 5%, usually 1 to 2%, of the weight and volume of the material used in each particular process.
Other techniques that have been applied include linkage to ion exchangers and absorption to a solid carrier. An example of such an application can be found in U.S. Pat. No. 4,699,882. However, the carrier used in this prior art technique is relatively expensive and the technique requires a large reactor.
The immobilization of enzymes for industrial use entails costs which are not necessarily associated with the used enzyme; they include costs caused by the construction of the process apparatus and the factory premises, carrier material acquisition (reacquisition) costs, cost of disposing of inactivated enzyme material, labor costs caused by emptying and filling reactors (or by the regeneration of the carrier) and secondary costs caused by the slowness of the reactors. As a consequence of long retention times, non-enzymatic, disadvantageous side reactions can often occur, particularly in the production of fructose.
Technically, cross-linking with glutaraldehyde has been of great importance in the immobilization of glucose isomerase. As is known, glutaraldehyde is approved by the FDA for the immobilization of enzymes to be used in food processing.
In addition, the scientific literature includes several examples of the cross-linking of crystalline enzymes by means of glutaraldehyde for basic research purposes. The structure of enzyme crystals is often so weak that the crystals do not withstand the ray beam used in X-ray diffraction studies; however, with glutaraldehyde they can often be stabilized for such purposes. Furthermore, crystals have been cross-linked with the purpose of studying stability and catalysis kinetics. In cases where the cross-linking of crystals has been successful, only glutaraldehyde has been used and the medium has consisted of a solution in which each particular enzyme is maintained in crystalline form. It appears that an insoluble crystal has been formed directly by a reaction between the glutaraldehyde and the enzyme protein. Quiocho and Richards (Proc. Natl. Acad. Sci. (USA) 52 (1964) p. 833 and Biochemistry 5 (1966) p. 4062) were the first to use glutaraldehyde in the cross-linking of carboxypeptidases. Bishop and Richards (J. Mol. Biol. 33 (1968) p. 415-421) have cross-linked crystalline beta lactoglobulin with a 1% aqueous solution of glutaraldehyde at room temperature. The crystals were used for studying the electrical properties of the enzyme. Haas (Biophysic. Journ. 8 (1968) p. 549-555) has cross-linked lysozyme crystals in the presence of a 4% sodium nitrate solution (pH 8), using a glutaraldehyde concentration of 12%.
Dyer, Phillips and Townsend (Thermochimica Acta 8 (1974) p. 456-464) have studied the thermostability of a crystalline carboxypeptidase cross-linked by glutaraldehyde. They have found that cross-linking leads to increased stability. Tuechsen and Ottesen (Carlsberg Res. commun. 42 (1977) p. 407-420) have studied the kinetic properties of a crystalline subtilisin cross-linked by glutaraldehyde in a sodium sulfate solution. With low-molecular substrates, the activity of the crystals was high whereas with high-molecular substrates (that could not diffuse into the crystals) the activity was low.
Wong et al. (Biochem. and Biophysic. Research Communications 80 (1978) p. 886-890) have cross-linked an acidic protease of microbial origin with glutaraldehyde in an ammonium sulfate solution. In the cross-linking, the presence of ammonium sulfate was regarded as a technical disadvantage.
Morozov and Morozova (Biopolymers 20 (1981) p. 451-467) have cross-linked crystalline lysozyme, hemoglobin and myoglobin using 2 to 6% glutaraldehyde solutions and a reaction time of 2 to 10 days at room temperature. Lee et al. (Bioorganic Chemistry 14 (1986) p. 202-210) have cross-linked crystals of alcohol dehydrogenase with glutaraldehyde in the presence of 2-methyl-2,4-pentanediol (25%).
It is often difficult to produce insoluble enzymes by means of glutaraldehyde, especially if the protein contains relatively little lysine. This problem is often circumvented by mixing into the enzyme a protein such as albumen which can be cross-linked to produce an insoluble form (G. B. Broun, Methods in Enzymology, 44 (1976) p. 263). The addition of such an inert foreign protein is not, however, possible when the enzyme to be cross-linked is crystalline. To date, there have been no means of cross-linking in cases where it is not possible to cross-link a crystal to insoluble form by means of glutaraldehyde only.